Genotyping and subtyping of Cryptosporidium spp. and Giardia duodenalis isolates from two wild rodent species in Gansu Province, China

Cryptosporidium spp. and Giardia duodenalis are commonly detected intestinal protozoa species in humans and animals, contributing to global gastroenteritis spread. The present study examined the prevalence and zoonotic potential of Cryptosporidium spp. and G. duodenalis in Himalayan marmots and Alashan ground squirrels in China's Qinghai-Tibetan Plateau area (QTPA) for the first time. Four hundred ninety-eight intestinal content samples were collected from five counties of QTPA of Gansu province, China. All samples were examined for Cryptosporidium spp. and G. duodenalis by PCR amplification. The resultant data were statistically analyzed by chi-square, Fisher's test and Bonferroni correction using SPSS software 25. 0. Cryptosporidium positive samples were further subtyped through analysis of the 60-kDa glycoprotein (gp60) gene sequence. A total of 11 and 8 samples were positive for Cryptosporidium spp. and G. duodenalis, respectively. Prevalence of Cryptosporidium spp. and G. duodenalis were 2.5% (10/399) and 1.5% (6/399) in Himalayan marmots, 1.0% (1/99) and 2.0% (2/99) in Alashan ground squirrels, respectively. Sequence analysis confirmed the presence of C. rubeyi (n = 2), ground squirrel genotype II (n = 7), chipmunk genotype V (n = 1) and horse genotype (n = 1). The horse genotype was further subtyped as novel subtype VIbA10. G. duodenalis zoonotic assemblages A (n = 1), B (n = 6), E (n = 1) were identified in the present study. This is the first study to identify Cryptosporidium spp. and G. duodenalis in Himalayan marmots and Alashan ground squirrels, suggesting the potential zoonotic transmission of the two pathogens in QTPA.

PCR amplification. Cryptosporidium spp. was detected by nested PCR amplification of the fragment (approximately 830 bp) of the small subunit (SSU) rRNA gene 22 . Subtyping of Cryptosporidium spp. was performed by sequence analysis of the 60 kDa glycoprotein (gp60) gene 23 . All the isolates of Cryptosporidiumpositive samples were selected for further sequence characterization via the actin gene and 70-kDa heat shock protein (HSP70) gene 61,62 . The assemblages of G. duodenalis were identified and subtyped by amplifying the β-giardin (bg), glutamate dehydrogenase (gdh) and triosephosphate isomerase (tpi) [24][25][26] . DNA of human-derived C. parvum and C. viatorum were used as positive controls in PCR tests to amplify the SSU rRNA, gp60, actin and HSP70 genes, respectively. Premiers and reaction conditions were shown in Supplementary Table S1. DNA of human-derived G. duodenalis was used as a positive control in PCR tests to amplify the bg, gdh and tpi genes. DNase-free water was used as a negative control in each PCR test. The secondary PCR products were visual- Obtained DNA sequences were aligned with reference sequences deposited in GenBank databases (http:// www. ncbi. nlm. nih. gov) using Clustal X (http:// www. clust al. org/) to determine the species/subtypes of Cryptosporidium spp. and assemblages of G. duodenalis. Phylogenetic analyses at the SSU rRNA, actin, HSP70 and gp60 gene loci were performed using the neighbor-joining model in MEGA 11 (http:// www. megas oftwa re. net). Bootstrap analysis was used to assess the robustness of the clusters using 1 000 replicates.
Statistical analysis. Differences in prevalence of Cryptosporidium spp. and G. duodenalis in Himalayan marmots and Alashan ground squirrels were compared among species and investigated area using the were processed with chi-square test, Fisher's exact test and pairwise comparisons used a Bonferroni correction to control for multiple testing. All the statistical analyses were performed using SPSS 25. 0 (SPSS Inc., New York, USA). Differences were considered significant at P < 0.05.
Cryptosporidium genotypes and subtypes. Based on sequence analysis of the SSU rRNA gene, a total four species/genotypes of Cryptosporidium spp. were identified out of 11 isolates, including C. rubeyi (n = 2), ground squirrel genotype II (n = 7), and chipmunk genotype V (n = 1) in Himalayan marmots, and horse genotype (n = 1) in Alashan ground squirrels. Cryptosporidium ground squirrel genotype II was dominant in Hima- www.nature.com/scientificreports/ layan marmots, accounting for 70.0% (7/10) of Cryptosporidium isolates. At the SSU rRNA gene locus, the two identical sequences of C. rubeyi shared the most significant identity (98.43%) with that of C. rubeyi (DQ295012) from California ground squirrels in the USA, with 13 base differences. Seven sequences of ground squirrel genotype II were identical and shared the most prominent similarity (98.28%) to that of the ground squirrel genotype II (KT027480) from black-tailed prairie dogs, with 14 base differences. The sequence of the chipmunk genotype V had 98.90% homology with that (MW521250) of the chipmunk genotype V from chinchillas in China, with nine base differences. The sequence of the horse genotype obtained in the present study had 100% homology with a sequence (MK775040) from a horse in China. The horse genotype isolate was further subtyped by sequence analysis of the gp60 gene. This subtype belonged to the VIb subtype family and was identified as VIbA10 (GenBank: MW531716). None of the two sequences of C. rubeyi were successfully amplified at the HSP70 gene locus but successfully amplified at the actin gene locus, and the two sequences were identical to each other, had 100% similarity with that of C. rubeyi (GenBank: KT027530) from black-tailed prairie dog. Meanwhile, two of seven isolates of ground squirrel genotype II were successfully amplified at the actin gene locus, and the two isolates shared the same sequence which had 97.68% similarity with that of ground squirrel genotype II (GenBank: KT027545) from black-tailed prairie dog in the USA. The HSP70 sequences have not been reported for ground squirrel genotype II. Three of seven isolates of ground squirrel genotype II were successfully amplified at the HSP70 gene locus and had 93.50% similarity with that of C. viatorum (GenBank: JX978274) from human in Guatemala. The sequence of chipmunk genotype V was only successfully amplified at the actin gene locus and shared 99.69% identity with that of chipmunk genotype V (MW521262) from chinchillas in China. Horse genotype was successfully amplified at the actin gene locus and shared 100% similarity with horse genotype (KU892571) isolated from humans of Kenya.
G. duodenalis assemblages. A total of eight G. duodenalis isolates were amplified and sequenced successfully in Himalayan marmots and Alashan ground squirrels in this study. Assemblages A, B and E were identified in one, four and one Himalayan marmot samples, respectively. Assemblage B was found in two Alashan ground squirrel samples. Meanwhile, assemblage B was observed to show a predominance (75.0%, 6/8) in the detected animals. The gdh and bg genes were successfully amplified in five samples-assemblages B (n = 4) and E (n = 1) and seven samples-assemblages A (n = 1), B (n = 5) and E (n = 1), respectively (Table 1). In this study, PCR amplification failed at the tpi locus.
At the gdh locus, two assemblage B sequences had 100% homology with beaver-derived assemblage B isolated (KM977648) from China. Another two different assemblage B sequences were 100% identical to golden monkeyderived assemblage B isolate (MK952602) from China, and one assemblage E sequence was 100% identical to a pig-derived assemblage E isolate (MK426742) from South Korea. At the bg locus, five assemblage B sequences shared 100% homology with squirrel monkey-derived assemblage B isolate (KJ888974) from China, one assemblage A sequence had 100% homology with human-derived assemblage A isolates (GQ329671) from Sweden and chipmunk-derived isolate (MF671918) from China, one assemblage E sequence (GenBank: MZ494459) shared the most considerable similarity (99.79%) to that (KY633473) from a Tibetan sheep in China, with only one base difference.

Discussion
In this study, the overall prevalence of Cryptosporidium spp. were 2.2% (11/498), with 2.5% in Himalayan marmots, and 1.0% in Alashan ground squirrels. There was no significant difference in the prevalence of Cryptosporidium spp. and G. duodenalis, and we will enlarge the research sample size for further verification. Other studies reported much higher prevalence of Cryptosporidium spp. in wild rodent species in China than this study, including in house mice (3.2%, 1/31), long-tailed rats ( www.nature.com/scientificreports/ ground squirrels, Belding's ground squirrels, and black-tailed prairie dogs 43,44 . Previously ground squirrel genotype II and chipmunk genotype V were only identified in black-tailed prairie dogs in the USA 43 and chinchillas in China 38 , respectively. Our identification of ground squirrel genotype II and chipmunk genotype V expanded the host range of the two genotypes. Horse genotype was initially isolated from a Przewalski wild horse at the Prague Zoo in the Czech Republic, and commonly detected in horses and donkeys, occasionally found in neonatal calves and hedgehogs 45,46 . Horse genotype has also been found in human patients with diarrhea in the UK and the USA, suggesting its zoonotic potential [47][48][49] . In the present study, the horse genotype was identified in rodents for the first time, indicating it has a broader range of host than initially anticipated. Horse genotype isolated from Alashan ground squirrels was further identified as novel subtype VIbA10. Currently, two subtype families are recognized within the Cryptosporidium horse genotype by sequence analysis targeting the gp60 gene: the VIa subtype family in animals (horses, donkeys and calves, etc.) and the VIb subtype family in humans and hedgehogs.  45 ; C. rubeyi in California ground squirrels (Spermophilus beecheyi) in the USA, Belding's ground squirrels (Spermophilus beldingi) and golden-mantled ground squirrels (Spermophilus lateralis) in the USA 44,53,54 ; ground squirrel genotype I and ground squirrel genotype III in thirteen-lined ground squirrels (Spermophilus tridecemlineatus) in USA 43 .
In this study, the overall prevalence of G. duodenalis were 1.6% (8/498), with 1.5% (6/399) for Himalayan marmots and 2.0% (2/99) for Alashan ground squirrels. This study reported much lower prevalence of G. duodenalis than other studies in wild rodent species in China: house mouse (3.2%, 1/31); Asian house rat (6.1%, 2/33); brown rat (6.6%, 11/168 and 9.3%, 33/355); pet chipmunks (8.6%, 24/279); bamboo rat ( (Table 3).   www.nature.com/scientificreports/ In this study, the sequences of amplicons from G. duodenalis-positive samples were determined to be assemblages A, B, and E, with assemblages B showing dominance in the detected animals. Assemblages A, B and E were identified in Himalayan marmots and assemblage B in Alashan ground squirrels. G. duodenalis assemblages in Himalayan marmots were richer than Alashan ground squirrels. As we know, in previous studies, G. duodenalis infections of Chinese rodents were reported to be caused by assemblages A, B and G 27,39,55-58 . Among them, assemblages A and B have a broad host range and are commonly found in humans 56 . Some recent studies in China www.nature.com/scientificreports/ also reported the occurrence of assemblage A in pet chipmunks, coypus and pet chinchillas, while assemblage B in bamboo rats, coypus and pet chinchillas [55][56][57][58] . These two assemblages were detected in this study suggest that Himalayan marmots and Alashan ground squirrels can play roles in the zoonotic dissemination of G. duodenalis. Assemblage E is commonly found in a range of hoofed livestocks and occasionally found in rodent species, and it has also been found in human cases, indicating that this assemblage is of zoonotic significance 59,60 .
In the investigated areas of QTPA, wild rodent species Himalayan marmots and Alashan ground squirrels have strong migration habits and often share pasture with humans, herbivorous animals and other wild animals. Results of this study suggest that these two wild rodent species may play a role in the transmission cycle of Cryptosporidium spp. oocysts and G. duodenalis cysts among humans, animals, water sources and fresh produce in QTPA grassland ecosystem.

Conclusion
This study examined the prevalence and zoonotic potential of Cryptosporidium spp. and G. duodenalis in Himalayan marmots and Alashan ground squirrels in the Qinghai-Tibetan Plateau area (QTPA) of China for the first time. Four Cryptosporidium species/genotypes were identified, including C. rubeyi, ground squirrel genotype II, chipmunk genotype V and horse genotype (novel subtype VIbA10). These two rodent species identified G. duodenalis zoonotic assemblages A, B, and E. The results expanded the host range of Cryptosporidium spp. and G. duodenalis, providing more information on the prevalence, epidemiology and genetic characterizations of the two pathogens in Himalayan marmots and Alashan ground squirrels. Further surveys are also required to understand the prevalence and transmission dynamics of the two pathogens.